Biomedical subjects

Ali A Yunus

Publications and source records attributed to Ali A Yunus.

3 recordsLinked to original sources

PubMed2006

Stat1 and SUMO modification.

Many proteins are known to undergo small ubiquitin-related modifier (SUMO) modification by an E1-, E2-, and E3-dependent ligation process. Recognition that protein inhibitor of activated signal transducers and activators of transcription (STATs) (PIAS) proteins are SUMO E3 ligases raised the possibility that STATs may also be regulated by SUMO modification. Consistent with this possibility, a SUMO-ylation consensus site (PsiKxE; Psi indicates hydrophobic residue, and x indicates any residue) was identified in Stat1 (ie, (702)IKTE(705)), but not in other STATs. Biochemical analysis confirmed that Stat1 K(703) could be SUMO modified in vitro. Mutation of this critical lysine (ie, Stat1(K703R)) yielded a protein that, when expressed in Stat1(-/-) mouse embryonic fibroblasts (MEFs), exhibited enhanced DNA binding and nuclear retention. This was associated with modest changes in transcriptional and antiviral activity. However, mutation of the second critical residue in the SUMO consensus site, E(705) (ie, Stat1(E705A)), yielded a protein with wild-type DNA binding, nuclear retention, and transcriptional and antiviral activity. Similar observations were made when these mutants were expressed in primary Stat1(-/-) macrophages. These observations suggest that although Stat1 can uniquely be SUMO-ylated in vitro, this modification is unlikely to play an important role in regulating Stat1 activity in vivo.

Animals↗

PubMed2006

Lysine activation and functional analysis of E2-mediated conjugation in the SUMO pathway.

E2 conjugating proteins that transfer ubiquitin and ubiquitin-like modifiers to substrate lysine residues must first activate the lysine nucleophile for conjugation. Genetic complementation revealed three side chains of the E2 Ubc9 that were crucial for normal growth. Kinetic analysis revealed modest binding defects but substantially lowered catalytic rates for these mutant alleles with respect to wild-type Ubc9. X-ray structures for wild-type and mutant human Ubc9-RanGAP1 complexes showed partial loss of contacts to the substrate lysine in mutant complexes. Computational analysis predicted pK perturbations for the substrate lysine, and Ubc9 mutations weakened pK suppression through improper side chain coordination. Biochemical studies with p53, RanGAP1 and the Nup358/RanBP2 E3 were used to determine rate constants and pK values, confirming both structural and computational predictions. It seems that Ubc9 uses an indirect mechanism to activate lysine for conjugation that may be conserved among E2 family members.

Amino Acid Sequence↗

PubMed2005

Purification and activity assays for Ubc9, the ubiquitin-conjugating enzyme for the small ubiquitin-like modifier SUMO.

The small ubiquitin-like modifier (SUMO) can be conjugated to lysine residues directly by the ubiquitin-conjugating protein Ubc9. SUMO conjugation can be catalyzed in vitro using only E1, Ubc9 (E2), mature SUMO, and ATP because Ubc9 directly recognizes consensus SUMO modification sites found in many identified targets of SUMO conjugation. This article describes methods to prepare Ubc9 and provides details for assay conditions used to evaluate E2 thioester formation and E2-mediated SUMO conjugation under single turnover and multiple turnover conditions. It also briefly describes parameters used to evaluate E3-mediated SUMO conjugation. Conservation of the SUMO conjugation apparatus from yeast to human has enabled in vivo assessment of human Ubc9 function through yeast complementation assays.

Blotting, Western↗